The major cause of death in many industrialized countries is atherosclerosis, a degenerative disease in which the walls of the arteries slowly become thickened by deposits of fatty material such as cholesterol. The deposits, or plaques, inhibit blood flow, often leading to heart attack or stroke. The personal and economic costs of atherosclerosis, and particularly of the form known as coronary artery disease (CAD), are vast. The economic cost of atherosclerosis has been estimated at 60 billion dollars annually.
Cholesterol is a building block for such hormones as estrogen and testosterone, as well as a fundamental structural component of animal cell membranes. When animal cells are growing and dividing, cholesterol required for new membrane material is transported to the site of growth via the blood. Cholesterol is transported in the blood in lipid-protein particles. The four major classes of such particles are the chylomicrons, the very low density lipoproteins (VLDL), the low density lipoproteins (LDL) and the high density lipoproteins (HDL).
Numerous studies have demonstrated that high concentrations of LDL in the blood plasma strongly correlate with increased risk of CAD. Thus, LDL is popularly called "the bad cholesterol". On the other hand, epidemiological and genetic studies have indicated both that high plasma concentrations of HDL correlate with protection against CAD, and that low plasma concentrations of HDL are involved in the development of atherosclerosis [Gordon et al., Am. J. Med. 62: 707-714 (1977); Stampfer et al., N. Engl. J. Med. 325: 373-381 (1991); Kuusi et al., Arteriosclerosis 7: 421-425 (1987); Moll et al., Am. J. Human. Genet. 44: 124-139 (1989); Hamsten et al., Atherosclerosis 60: 199-208 (1986)]. Thus, the popular nickname for HDL is "the good cholesterol".
The major protein component of HDL is apolipoprotein (apo) AI, which is believed to promote the process of "reverse cholesterol transport" [Gotto et al., Methods Enzymol. 128: 3-41 (1986); Miller et al., Nature (London) 314: 109-111 (1985); Glomset, Adv. Intern. Med. 25: 91-116 (1980)]. In this process, excess cholesterol is liberated from the peripheral tissues and carried, via HDL, to the liver for degradation. In addition, apo AI acts as a cofactor for the enzyme lecithin-cholesterol acyltransferase (LCAT), which is also involved in reverse cholesterol transport [Gotto et al., Methods Enzymol. 128: 3-41 (1986); Miller et al., Nature (London) 314: 109-111 (1985); Glomset, Adv. Intern. Med. 25: 91-116 (1980)]. Further evidence that apo AI is a strong negative factor for atherosclerosis comes from experiments in which transgenic mice carrying the human apo AI gene were fed a high fat diet. Here, expression of the apo AI transgene and the resulting high levels of human apo AI in the animals' blood appeared to protect against development of fatty streak lesions [Rubin et al., Nature (London) 353: 265-267 (1991)]. By extension, if the expression of the apo AI gene in humans could be increased, it would likely protect against deposition of atherosclerotic plaques.
The foregoing and other studies indicate that the regulation of apo AI gene expression is extremely important in controlling the development of CAD. In mammals, apo AI expression is tissue-specific, with synthesis taking place primarily in the liver and intestine [Gotto et al., Methods Enzymol. 128: 3-41 (1986)]. Presumably, tissue-specific factors are responsible for this limited expression pattern.
A number of factors have been reported to modulate plasma levels of apo AI and/or HDL cholesterol. These include diet [Knuiman et al., Arteriosclerosis 7: 612-619 (1987)], exercise [Berg et al., Clin. Chim. Acta 161: 165-171 (1986)], sex hormones [Tam et al., J. Biol. Chem. 260: 1670-1675 (1985)], thyroid hormone [Davidson et al., J. Lipid Res. 29: 1511-1522 (1988)], ethanol [Tam, Alcohol. Clin. Exp. Res. 16: 1021-1028 (1992)] and temporal factors during development [Haddad et al., J. Biol. Chem. 26: 13268-13277 (1986); Staels et al., J. Lipid Res. 30: 1137-1145 (1989)]. In addition, clinical studies have shown that there is direct relationship between the levels of cytochrome P450 enzymes in the liver and the levels of HDL and apo AI in the blood [Luoma et al., Acta Med. Scand. 214: 103-109 (1983); Luoma, Pharm. & Toxic. 65: 243-249 (1988)]. Cytochrome P450 enzymes function in the detoxification of aromatic compounds and in bile acid and vitamin D metabolism. The nature of the relationship between hepatic cytochrome P450 levels and plasma apo AI levels is not fully understood.
Clinically, a number of drugs have been used to reduce the risk and/or progression of CAD by altering lipoprotein metabolism. Gemfibrozil (Lopid.TM.), manufactured by Parke Davis of Morris Plains, N.J., is one of the most extensively studied pharmaceutical agents for the treatment of hyperlipidemia [Manninen et al., Circulation 85: 37-45 (1992): Brown, Am. J. Cardiol. 66: 11 A-15A (1990)]. In patients treated with gemfibrozil, both total and LDL cholesterol levels in the plasma are reduced, whereas HDL cholesterol levels are markedly raised. In the classic Helsinki Heart Study [Frick et al., N. Engl. J. Med. 317: 1237-1245 (1987)], the administration of gemfibrozil over a five year period to middle-aged men who were at high risk because of abnormal concentrations of blood lipids resulted in a 34% reduction in coronary disease, relative to a similar group of men who received only a placebo. At least part of this effect may be ascribed to the action of gemfibrozil in significantly raising HDL concentrations.
One of the present inventors has previously reported that the exposure of two human hepatoma cell lines, HepG2 and Hep3B, to gemfibrozil results in a two-fold increase in the level of apo AI mRNA [Tam, Atherosclerosis 91: 51-61 (1991)]. Prior to the invention described below, the mechanism of gemfibrozil action in regard to apo AI levels was unknown.
The human apo AI gene is located on the long arm of chromosome 11. The DNA sequence of this gene is identified in Karathanasis et al., Nature (London) 304: 371-373 (1983); Breslow et al., Proc. Nat. Acad. Sci. USA 79: 6861-6865 (1982); and GenBank accession no. M20656. Cis- and trans-acting elements involved in the regulation of transcription of the apo AI gene have been studied by several groups [Sastry et al., Mol. Cell. Biol. 8: 605-614 (1988); Widom et al., Mol. Cell. Biol. 11: 677-687 (1991); Papazafiri et al., J. Biol. Chem. 266: 5790-5797 (1991); Pagani et al, J. Lipid Res. 31: 1371-1377 (1990); Smith et al., J. Clin. Invest. 89: 1796-1800 (1992); Sigurdsson et al., Arteriosclerosis and Thrombosis 12: 1017-1022 (1992); Tuteja et al., FEBS Letters 304: 98-101 (1992); Jeenah et al., Mol. Biol. Med. 7: 233-241(1990)]. FIG. 1 shows in block form the organization of the 5' flanking region of the human apo AI gene, according to the results obtained by such groups.
In a first study conducted by Karathanasis and co-workers [Sastry et al., Mol. Cell. Biol. 8: 605-614 (1988)], transient transfection assays were used to identify a liver-specific enhancer element located between nucleotides -256 to -41 upstream of the transcription start site (+1) of the human apo AI gene. Evidence was presented that this DNA region contains regulatory elements that are necessary and sufficient for maximal expression of the gene in the human hepatoma cell line HepG2.
In a further study [Widom et al., Mol. Cell. Biol. 11: 677-687 (1991)], the same group used gel mobility shift and DNase I footprinting assays to identify three distinct sites within the enhancer, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), to which transcriptional factors present in HepG2 cells specifically bind. These regions are indicated in FIG. 1 by A*, B* and C*. The researchers found that binding of a factor to a single site in the absence of binding to the others was not sufficient for gene expression. Binding of factors to any two of the sites resulted in low levels of expression. Binding to all three sites was necessary for maximal gene expression. It was hypothesized that protein-protein interactions between the bound transcription factors provided this synergistic effect.
In another fine structure study [Papazafiri et al., J. Biol. Chem. 266: 5790-5797 (1991)], Zannis and co-workers identified four DNA regions proximal to the human apo AI gene that were protected against digestion by DNAse I by the binding of factors contained in rat liver nuclear extracts. The protected regions were designated A (-22 to +17), B (-128 to -77), C (-175 to -148) and D (-220 to -190), as shown in FIG. 1. The rat nuclear proteins that bound to the region -220 to -148 (i.e., regions D and C) were identified using in vitro mutagenesis, DNA binding assays and protein fractionation. Both positive and negative regulators were identified.
At least three different groups have studied the effect on apo AI levels of a G to A transition variously identified as occurring at -78 [Pagani et al., J. Lipid Res. 31: 1371-1377 (1990); Tuteja et al., FEBS Letters 304: 98-101 (1992)], at -76 [Smith et al., J. Clin. Invest. 89: 1796-1800 (1992)] and at -75 [Jeenah et al., Mol. Biol. Med. 7: 233-241(1990); Sigurdsson et al., Arteriosclerosis and Thrombosis 12: 1017-1022 (1992)] relative to the start of transcription of the human apo AI gene. Despite the discrepancy in numbering, the studies seem to concern the same point mutation, in which an Msp I restriction site (CCGG) is destroyed by the substitution of A at the position of the first G. This substitution, which is at the border of region B identified by the Zannis group, occurs naturally in the human population.
The first of these groups, Baralle and co-workers, reported that the presence of the A allele was associated with high HDL cholesterol and apo AI levels in women, but not in men [Pagani et al., J. Lipid Res. 31: 1371-1377 (1990)]. They noted that the polymorphism occurred in a 51 bp GC-rich region containing an inverted repeat composed of two 14/15 bp elements. The homology and self-complementarity of the inverted repeats was disrupted when a G, instead of an A, was present at the -78 position. In transient transfection assays using a DNA fragment from -330 to +1, the A allele demonstrated about two-fold higher activity than the G allele [Tuteja et al., FEBS Letters 304: 98-101 (1992)]. However, when a larger DNA fragment from -1469 to +397 was used, the two alleles displayed similar transcriptional levels.
A second group, Breslow and co-workers, reported that patients that were G/A heterozygotes at this position displayed significantly lower apo AI production rates than G/G homozygotes [Smith et al., J. Clin. Invest. 89: 1796-1800 (1992)]. (In a human subject, the apo AI production rate takes into account not only the level of apo AI synthesis, but also its levels of intracellular transport, secretion and plasma clearance.) Despite their different production rates, no difference was found in HDL cholesterol or apo AI levels between the two groups of patients. When the expression levels of the two alleles were compared in transient transfection assays employing a 325 bp fragment of the apo AI gene, the expression level of the A allele was found to be only approximately 68% of the expression level of the G allele. These results are in direct contrast with those of the Baralle group. However, like the Baralle group, the Breslow group also explained their results in terms of the effect of the point mutation on the inverted repeats. They speculated that the increased complementarity of the inverted repeats by the presence of an A at -76 might allow for the formation of a DNA secondary structure that might interfere with protein-protein interactions of the transcriptional apparatus.
A third group, Humphries and co-workers, reported that men having high plasma apo AI levels carried the A allele more than twice as often as men with lower plasma apo AI levels [Jeenah et al., Mol. Biol. Med. 7: 233-241(1990)]. In addition, men carrying the A allele displayed significantly higher HDL cholesterol levels. Thus, this work contrasts with both the Baralle group's findings, in which only women were affected, and the Breslows group's findings, in which no difference in apo AI and HDL cholesterol levels were identified between carriers of the two alleles. In a subsequent study, the Humphries group went on to report that the protection against risk of CAD that had been observed for men carrying the A allele was abolished if the men smoked [Sigurdsson et al., Arteriosclerosis and Thrombosis 12: 1017-1022 (1992)]. That is, male smokers carrying the A allele were found to have approximately the same apo AI and HDL cholesterol levels as men carrying the G allele.
Thus, although a great deal of work has been done to date on the regulation of expression of the human apo AI gene, much clarification is still required. The various cis- and trans-acting factors involved and the nature of their interactions need to be identified and elucidated. In particular, the mechanisms by which various drugs, for example, gemfibrozil, influence apo AI expression are heretofore unknown. Given the protection that high plasma apo AI levels provide against CAD, it would be extremely desirable to understand how a particular drug could increase apo AI expression. A convenient method and tools for screening for such a drug would also be extremely desirable.